The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene (in a foreign DNA) at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps that include extensive genetic characterization, breeding, and evaluation in field trials.
The term “rapeseed” covers every seed of the Brassica species. Brassica is cultivated from China and India to Finland and Canada as one of the most valuable oil crops. Most Brassica types belong to the family of Cruciferae. They originated as a diploid species having aneuploid chromosome numbers ranging from 7 (Brassica fruticulosa) to 12 (Sinapsis alba).
The most extensively grown Brassica species in Canada is known as turnip rape, or Brassica campestris (aa, n=10). Brassica oleracea (cc, n=9) has diversified in recent evolutionary history into at least six major horticultural types, including broccoli, cauliflower and cabbage. Brassica nigra (bb, n=8) or black mustard is a less important crop commercially and is mainly known for its seeds from which mustard was originally made. From these basic types, amphiploid hybrids have been derived in more recent evolutionary stages by intercrossing. The most important of these are Brassica napus (aacc), of which the winter types provide the highest rapeseed yields in northern Europe and Brassica juncea (aabb) or brown mustard that is one of the major oil crops of the Indian sub-continent.
Though intercrossing between different Brassica species (particularly those with compatible genomes) is possible and often done for breeding purposes, not all traits (or genes) will be able to be transferred from one species to the other or, when transferred to a different species, will not retain identical characteristics (or expression patterns). Thus, a genetic locus conferring optimal expression of a natural or chimeric gene in one Brassica species, will not necessarily have the same effect in another.
Brassica species are bisexual and typically 60-70% self-pollinated. The production of hybrids and introduction of genetic variation as a basis for selection was traditionally dependent on the adaptation of natural occurring phenomena such as self-incompatibility and cytoplasmic male-sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides are not widely applied in Brassica breeding due to their limited practicability and high cost respectively.
Transgenic methods have been developed for the production of male or female-sterile plants, which provide interesting alternatives to the traditional techniques.
EP 0,344,029 describes a system for obtaining nuclear male-sterility where plants are transformed with a male-sterility gene, comprising for example a DNA encoding a barnase molecule under the control of a tapetum specific promoter TA29, which when incorporated into a plant ensures selective destruction of tapetum cells. Transformation of tobacco and oilseed rape plants with such a gene resulted in plants in which pollen formation was completely prevented. Mariani et al. (1990) Nature 347:737-741.
Cytochemical and histochemical analysis of anther development of B. napus plants comprising the chimeric PTA29:barnase gene is described by De Block and De Brouwer ((1993) Planta 189:218-225).
To restore fertility in the progeny of a male-sterile plant, a system was developed where the male-sterile plant is crossed with a transgenic plant carrying a fertility-restorer gene, which when expressed is capable of inhibiting or preventing the activity of the male-sterility gene. U.S. Pat. Nos. 5,689,041; and 5,792,929; and De Block and De Brouwer (1993).
The use of coregulating genes in the production of male-sterile plants to increase the frequency of transformants having good agronomic performance is described in WO 96/26283. Typically, when the sterility DNA encodes a barnase, the coregulating DNA will encode a barstar.
Successful genetic transformation of Brassica species has been obtained by a number of methods including Agrobacterium infection (as described, for example in EP 0,116,718 and EP 0,270,882), microprojectile bombardment (as described, for example by Chen et al. (1994) Theor. Appl. Genet. 88:187-192) and direct DNA uptake into protoplasts (as described, for example by De Block et al. (1989) Plant Physiol. 914:694-701; and Poulsen (1996) Plant Breeding 115:209-225).
However, the foregoing documents fail to teach or suggest the present invention.